P988: LACTATE RESHAPES TUMOR MICROENVIRONMENT AND METABOLIC PROFILE IN MYELOFIBROSIS
Authors: S. Giallongo, D. Tibullo, M. Spampinato, C. Giallongo, E. La Spina, L. Longhitano, A. Romano, I. Dulcamare, A. Barbato, G. Scandura, A. M. Amorini, G. Lazzarino, T. Zuppelli, R. Caltabiano, G. Li Volti, F. Di Raimondo, G. A. M. Palumbo
Journal: HemaSphere (2022)

Background: Primary myelofibrosis (PMF) is myeloproliferative neoplasm depicted by bone marrow (BM) fibrosis, ineffective clonal hematopoiesis, and splenomegaly. These outcomes are related with changes in bone marrow microenvironment, sustaining cancer glycolytic metabolism, fibrosis, immune evasion, and thrombotic events. Lactate, a glycolysis byproduct, may represent the driving-force towards the acquisition of these features. For this reason, we investigated PMF CD34+ cells lactate production as factor prompting the metabolic rewire of BM niche. Aims: In this work we aimed to target lactate metabolism as stricking factor improving PMF proliferative features. Methods: Primary mesenchymal stem cells (MSCs) and HS-5 proliferation assays were performed in vitro. Protein expression level was assessed using immunoblot and immunohistochemical assays. Moreover, Cytokines were also investigated ex vivo, using Multiplex immunobead assay technology. These results were also mirrored in vivo on TPOhigh Zebrafish (Danio rerio) model. Results: The monocarboxylate transporters (MCT-1 and -4), orchestrating lactate intake, were significatively upregulated in CD34+ cells and sera derived from MF-patients compared to healthy donor. MF-patients are characterized by increased percentage of circulating immunosuppressive cells. Incubation of peripheral blood mononucleated cells (PBMNCs) with MF-sera increased Treg and Myeloid-derived suppressor cells (M-MDSCs) expansion. This effect was disrupted by addition of MCT1 inhibitor AZD3965. The role of lactate on BM niche was evaluated by culturing healthy primary MSCs. Lactate (20 mM) and MF-sera exposition induced extracellular matrix (ECM) reorganization as supported by increased level of metalloproteases (MMP9, MMP2) together with reticulin and collagen deposition. Moreover, lactate induces calcium accumulation and soluble osteogenic molecular signals production (i.e. osteoprotegerin, osteonectin). On the epigenetic side, lactate treatment reshapes chromatin architecture eventually affecting histones lactylation, acetylation and methylation levels. These features were rescued upon AZD3965 treatment. Cancer-associated fibroblast (CAF) play an outstanding role in cancer development by ECM remodeling.Upon lactate exposure, expression of CAF markers α-SMA, FAP1 and TGFβ were upregulated in MSCs. The effect was reverted by AZD3965 treatment. The data achieved in vivo, and ex vivo were corroborated in a new pre-clinical TPOhigh Zebrafish model eventually showing increased Whole Kidney Marrow lactate. Summary/Conclusion: In this work we report MCT1 inhibition as a strategy to treat invasive and metastatic cancers. Our results corroborate the idea that targeting lactate metabolism may represent a promising path to counteract inflammation, osteosclerosis and fibrosis in PMF patients.